a molecular network for de novo generation of the apical ......to form epithelial organs cells must...

ART ICLES

A molecular network for de novo generation of the apical surface and lumenDavid M. Bryant1,5, Anirban Datta1,5, Alejo E. Rodríguez-Fraticelli3, Johan Peränen4, Fernando Martín-Belmonte3 and Keith E. Mostov1,2,6

To form epithelial organs cells must polarize and generate de novo an apical domain and lumen. Epithelial polarization is regulated by polarity complexes that are hypothesized to direct downstream events, such as polarized membrane traffic, although this interconnection is not well understood. We have found that Rab11a regulates apical traffic and lumen formation through the Rab guanine nucleotide exchange factor (GEF), Rabin8, and its target, Rab8a. Rab8a and Rab11a function through the exocyst to target Par3 to the apical surface, and control apical Cdc42 activation through the Cdc42 GEF, Tuba. These components assemble at a transient apical membrane initiation site to form the lumen. This Rab11a-directed network directs Cdc42-dependent apical exocytosis during lumen formation, revealing an interaction between the machineries of vesicular transport and polarization.

Most internal epithelial organs consist of a monolayer of polarized epithe-lial cells surrounding a central lumen. Polarization requires the interac-tion of the signalling complexes and scaffolds that define cortical domains with membrane-sorting machinery1. In yeast, traffic from the trans-Golgi network to the cell surface is regulated by Ypt32p and Sec4p (ref. 2), homo-logues of mammalian Rab11 and Rab8, respectively. Ypt32p recruits Sec2p (homologue of mammalian Rabin8), a GEF for Sec4p. Sec2p and Sec4p in turn interact with the exocyst, which docks vesicles to the cell surface3.

Definition of cortical domains in metazoa involves a complex of Par3, Par6, atypical protein kinase C (aPKC) and the GTPase, Cdc42 (ref. 4). This complex is a master regulator of polarity, conventionally depicted upstream of membrane-trafficking machinery. How this complex inter-faces with membrane transport is poorly understood.

Here, we show a molecular mechanism for lumen and apical surface formation, linking Rab8a and Rab11a, the exocyst, annexin2, Cdc42 and its GEF Tuba, and the Par3–aPKC complex. This pathway shows how the membrane traffic and cortical polarity machineries cooperate to generate the apical surface and lumen de novo.

RESULTS Apical polarization during lumen formationOn plating into 3D culture, individual MDCK (Madin-Darby canine kid-ney) cells proliferate and assemble into cyst structures—a polarized spherical monolayer surrounding a central lumen. Lumenogenesis requires the api-cal membrane determinant gp135/podocalyxin5 (PCX in figures). Initially, podocalyxin is localized at the extracellular matrix-contacting surface in MDCK aggregates (Fig. 1a, 12 h and Supplementary Information, Fig. S1a),

before polarity inversion occurs, with β-catenin and Na+/K+-ATPase at cell–cell junctions and podocalyxin now at the lumen (Fig. 1a, 24–48 h; arrows and Supplementary Information, Fig. S1d)6,7. Lumens start to form at a site termed the ‘pre-apical patch’ (PAP), where opposing plasma membranes are separated, but the podocalyxin signal does not give an optically resolvable lumen by confocal microscopy7. The lumen can only be visualized after expansion and further separation of apical membranes (Fig. 1b). Conversely, apical proteins syntaxin-3 and GFP–CNT1 (green fluorescent protein fused to concentrative nucleoside transporter-1) label the entire surface before concentrating at the lumen (Supplementary Information, Fig. S1b, c, e, f)5.

Binding of antibodies to GFP–VSVG–podocalyxin (VSVG; vesicular stomatis viral G protein) at the periphery of cysts, followed by incubation to allow lumenogenesis, revealed that podocalyxin at the PAP (arrow-heads) and in vesicles (arrows) is at least partially derived from transcy-tosed peripheral podocalyxin (Supplementary Information, Fig. S1g).

Transcytosis of podocalyxin to the luminal surface to establish the PAP represents formation of apical–basal polarization. We therefore exam-ined localization of select polarity (Par3–aPKC), trafficking (exocyst complex; Sec8–Sec10–Sec15A), and junctional (occludin) proteins dur-ing lumen initiation. Strikingly, although these proteins showed differing localizations before lumen formation, all converged transiently during lumen initiation (Fig. 1c–k and Table 1). When GFP–podocalyxin was peripheral, Par3 and Sec8 co-localized in puncta at the edge of cell–cell contacts (Fig. 1c; arrowheads). When GFP–podocalyxin was internalized and transcytosed, some Par3 and Sec8 concentrated at the first detectable site of GFP–podocalyxin delivery to the nascent apical surface (Fig. 1d; arrow). We term this the apical membrane initiation site (AMIS). Later,

1Department of Anatomy, University of California, San Francisco, CA 94143‑2140, USA. 2Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143‑2140, USA. 3Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Madrid 28049, Spain. 4Institute of Biotechnology, Viikinkaari 9, University of Helsinki, FIN‑00014 Helsinki, Finland. 5These authors contributed equally to this work. 6Correspondence should be addressed to K.E.M. ([email protected]).

Received 16 February 2010; accepted 30 July 2010; published online 03 October 2010; DOI: 10.1038/ncb2106

nature cell biology VOLUME 12 | NUMBER 11 | NOVEMBER 2010 1035

© 20 Macmillan Publishers Limited. All rights reserved10

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Par3 and Sec8 enriched at the tight junction (Fig. 1e; arrowheads). We define early apical structures where several tight-junction markers have become distinctly localized from podocalyxin as the PAP7, compared with the AMIS that forms earlier, where tight-junction markers and podocalyxin cannot be resolved by confocal microscopy.

In contrast, Sec10 and occludin initially localized along the entire cell–cell contact in early aggregates, which had peripheral podocalyxin (Supplementary Information, Fig. S2a and data not shown). Podocalyxin delivered to the AMIS partially overlapped with Sec10 and occludin (Fig. 1f and Supplementary Information, Fig. S2b; arrows). Although occludin remained along the entire contact, Sec10 condensed toward the AMIS. As the lumen expanded, Sec10 and occludin enriched at the tight junction (Fig. 1g, h and Supplementary Information, Fig. S2c–f), although some occludin remained along cell–cell contacts (Fig. 1h).

aPKC follows yet a different pattern, with distinct pools initially localized with peripheral podocalyxin (arrowheads) and the AMIS (Fig. 1i; arrows), before enriching at PAP edges and finally the tight junction and lumen (Fig. 1j, k; arrowheads). These data show the complex movement of traffick-ing and cortical polarity proteins that converge transiently at the AMIS.

The Rab8 and Rab11 GTPase families direct lumen initiationWe examined AMIS and lumen formation on perturbation of select Rab GTPases involved in apical, basolateral or junctional trafficking (Fig. 2a–f

and Supplementary Information, Fig. S3f–l). In contrast to control cysts with a single lumen, apical podocalyxin and basolateral β-catenin, knock-down of Rab8 (Rab8a/Rab8b) and Rab11 (Rab11a/ Rab25, but not Rab11b) family members significantly decreased single lumenogenesis (Fig. 2f) so that cysts had multiple lumens and accumulated podocalyxin in vesicles (Fig. 2b–e; arrowheads and data not shown) close to the cell surface (marked by β-catenin). For single lumen-perturbing knockdowns, phenotypes were confirmed using additional shRNAs (Supplementary Information, Fig. S3l), and additional cargoes (Supplementary Information, Fig. S3a–c). Perturbation of Rab10, Rab11b, Rab13 and Rab14 did not markedly perturb lumenogenesis, and were not investigated further (Fig. 2f).

The Rab11 family regulates transcytosis8 and lumenogenesis in diverse systems9–11. GFP–Rab11a localized to vesicles underlying the AMIS (marked by Par3; arrow in Fig. 2g), then remained on subapical vesicles once lumens expanded (Fig. 2h and Table 1). Podocalyxin transcytosed to the AMIS through Rab11a-positive vesicles. When podocalyxin was peripheral (Fig. 2i; arrow), GFP–Rab11a localized to juxtanuclear and peripheral vesicles (Fig. 2i; white and yellow arrowheads, respectively). On internalization, podocalyxin localized to GFP–Rab11a-positive vesicles (Fig. 2j; arrow-heads), then both were delivered to the cyst interior (Fig. 2k; arrowheads). Here, regions of podocalyxin devoid of GFP–Rab11a began to emerge (Fig. 2k–m; arrows), representing podocalyxin surface delivery. Similarly, immunoglobulin A (IgA) transcytosed to the PAP (Supplementary

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Figure 1 Characterization of lumen initiation in MDCK cysts. (a) Representative immunofluorescence confocal microscopy images showing development of polarity in MDCK cysts incubated with antibodies against the indicated proteins, and Hoescht (to visualize nuclei). Arrow at 24 h after plating indicates polarity inversion, with podocalyxin now at the cyst interior and formation of a PAP. Arrow at 48 h after plating indicates the opening of the luminal space. (b) Schematic representation of cyst development from a. Black lines, plasma membrane; red lines, apical surface; blue, nuclei. (c–k) Transient localization of polarity and trafficking machinery to an apical membrane initiation site (AMIS) in representative MDCK cysts at different

stages during lumen initiation. Bottom: higher‑magnification images of the indicated regions showing localization of individual proteins (c–e), or in f–k individual proteins (left, middle) and a merge of these two images (right). Arrowheads in c–e indicate Sec8 and Par3 localization. Arrow in d indicates co‑localization of Sec8, Par3 and GFP–podocalyxin (green) at the AMIS. Arrows in f indicate co‑localization of podocalyxin and occludin at the AMIS; arrowheads in g and h indicate localization of occludin at the edges of the lumen (g) and at the tight junctions (h). In i arrowheads indicate peripheral podocalyxin and aPKC. Arrows in i indicate localization of aPKC at the AMIS. In j–k arrowheads indicate aPKC at tight junction regions. Scale bars, 20 μm.

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Information, Fig. S1h). As the lumen expanded, GFP–Rab11a clustered underneath the apical surface (Fig. 2l, m). Notably, overexpression of GFP–Rab11a (wild-type or an activated Q70L substitution mutant) increased single lumenogenesis, whereas dominant negative GFP–Rab11aS25N attenu-ated single lumenogenesis and accumulated podocalyxin intracellularly (Supplementary Information, Fig. S4a; data not shown). Thus, Rab11a promotes transcytosis to the AMIS and single lumenogenesis.

Rab8 family GTPases were also required for single lumen forma-tion (Fig. 2b, c, f), and Rab8a localized to transcytosing podocalyxin vesicles and the AMIS7 (Table 1 and data not shown). Knockdown of Rab11a resulted in upregulation of Rab8a, and vice versa (Supplementary Information, Fig. S3f, g), suggesting compensation or cooperation between Rab11 and Rab8 families. Therefore, we knocked down Rab8 and Rab11 family members, alone or in combination (Supplementary Information, Fig. S4b). Of tested combinations, co-knockdown of Rab8a and Rab8b, with or without Rab11a knockdown, most markedly reduced single lumenogenesis. This suggests that the Rab8 family may function downstream of Rab11a. Accordingly, Rab8a knockdown blocked the increase in single lumenogenesis induced by GFP–Rab11aQ70L expression (Supplementary Information, Fig. S4c). These data are consistent with the hypothesis that the Rab8 family functions, at least in part, downstream of Rab11a during apical transport and lumenogenesis, although the precise interaction between these Rab proteins may be more complex.

Regulation of Rab8 during lumenogenesisRab11 binds to the Rab GEF Rabin8 and stimulates its activity towards Rab8 (ref. 12). We reasoned that Rab11a may control subapical Rabin8–Rab8 targeting. In control cysts, a small pool of Rabin8, and to a lesser extent Rab8a, localized to dispersed puncta, with some clustered subapically (Fig. 3a; arrows). Expression of GFP–Rab11aWT, but not GFP–Rab11aS25N, strongly enhanced recruitment of Rabin8 and Rab8a to Rab11a-positive subapical vesicles (Fig. 3a; arrowheads). Similarly to endogenous Rab8a (Fig. 3a), GFP–Rab8aWT was cytoplasmic and in subapical vesicles, the latter of which was enhanced on activated GFP–Rab8aQ67L expression (Supplementary Information, Fig. S4f). Thus, active Rab11a recruits active Rab8a to subapical vesicles, probably through Rabin8.

In western blots of MDCK lysates, Rabin8 was identified in two bands cor-responding to its α and β isoforms: both possess the Rab11-binding region12 (Fig. 3b and Supplementary Information, Fig. S5a). On Rabin8α knockdown some podocalyxin accumulated in vesicles (Fig. 3c; arrowheads), and there was also a small (but statistically significant) decrease in the proportion of cysts with single lumens (Fig. 3e). This effect is only modest probably because

of compensatory upregulation of Rabin8β observed on Rabin8α knockdown (Fig. 3b). Dual Rabin8α and Rabin8β knockdown caused cell death, preclud-ing further analysis (data not shown). In cysts with endogenous Rabin8α knockdown, expression of RNAi-resistant GFP–hRabin8αWT (GFP-tagged human Rabin8α), which localized to the luminal region (arrows), restored single lumenogenesis and podocalyxin localization (Fig. 3d, e). Conversely, Rabin8α GEF-domain mutants (Supplementary Information, Fig. S5a–c) further decreased single lumenogenesis and co-accumulated with podo-calyxin on vesicles (Fig. 3d, e; arrowheads) beneath the surface marked by F-actin. Similarly, overexpression of GFP–TBC1D30WT, a GAP (GTPase-activating protein) specific to the Rab8 family13, but not GAP-deficient GFP–TBC1D30R140A, perturbed single lumenogenesis (Supplementary Information, Fig. S5d, e). These data suggest that a Rab11a–Rabin8α–Rab8a cascade, inhibited by TBC1D30, is part of a regulatory module that governs apical transport and lumenogenesis.

The exocyst and Par3–aPKC complexes regulate apical polarizationRab8a and Rab11a interact with the Sec15 exocyst subunit14, in turn linking to Sec10 and other subunits as part of a chain tethering vesicles to the basolateral3 and apical membranes15. The exocyst also interacts with the Par3–aPKC complex16,17. As these factors converge at the AMIS, we examined their requirement in apical traffic and single lumenogenesis.

In contrast to control cysts with apical podocalyxin and basolateral β-catenin (Fig. 4a), Sec15A knockdown (Supplementary Information, Fig. S3m) resulted in accumulation of podocalyxin in prominent GFP–Rab11a-positive vesicles close to the apical surface (Fig. 4b; arrows, 4c; arrowheads). This Rab11a compartment seemed expanded relative to control cysts (compare Fig. 2m). Additionally, cysts were defective in apical polarization, mistargeting apical cargo to regions of cell–cell con-tact (Fig. 4b and Supplementary Information, Fig. S3d). Accordingly, Sec15A knockdown caused almost complete loss of single lumenogenesis (Fig. 4d). Similarly, Sec10 knockdown decreased single lumenogenesis and caused vesicular accumulation of podocalyxin (Supplementary Information, Fig. S2g–i). Thus, the exocyst regulates podocalyxin trans-port from Rab11a-positive vesicles to the forming apical surface.

We also examined the role of the exocyst on Par3 transport and AMIS formation. In control cysts, Par3 localized to tight junctions (Fig. 4e; arrows). In Sec15A knockdown cysts, Par3 showed varying, though always abnormal, localization. In regions where a PAP formed, Par3 was recruited to the sur-face (Fig. 4f). However, in regions of vesicular podocalyxin accumulation, Par3 failed to be recruited to the surface and an AMIS was undetectable

Table 1 Distribution of trafficking and polarity proteins during lumen initiation and expansion.

Lumen Stage

Protein AMIS PAP Open Lumen

Occludin Cell–cell contact Cell–cell contact Cell–cell contact/tight junction

Par3 AMIS PAP Tight junction

aPKC AMIS/peripheral PAP Tight junction/luminal

Podocalyxin Vesicles/peripheral PAP Lumen

Rab8a Vesicles Subapical vesicles Subapical vesicles

Rab11a Vesicles Subapical vesicles Subapical vesicles

Sec8 AMIS PAP Tight junction

Sec10 Cell–cell contact PAP Tight junction

Sec15A Vesicles Subapical vesicles Subapical vesicles



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(Fig. 4f; arrowhead). In cysts with endogenous Sec15A knockdown, expres-sion of RNAi-resistant GFP–Sec15AWT, which localized to subapical vesicles (Fig. 4g), rescued single lumenogenesis, surface delivery of podocalyxin and Par3 localization (that is, at tight junctions once lumens had formed).

To test the role of exocyst coupling to Rabs, we used GFP–Sec15AN691A, a mutant that does not bind to Rab11a15. This mutant was completely unable to rescue the trafficking and single lumenogenesis defects caused

by knockdown of endogenous Sec15A (Fig. 4h). Thus, coupling of exo-cyst to Rab8a or Rab11a is required for surface targeting of podocalyxin and Par3 to the AMIS.

Similar to exocyst knockdown, Par3 knockdown also resulted in intrac-ellular podocalyxin accumulation close to the surface marked by β-catenin (Fig. 4i; arrows), in vesicles co-labelled for GFP–Rab11a (Fig. 4j; arrows), and a strong disruption of single lumenogenesis (Fig. 4l). Par3 knockdown

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Figure 2 The Rab8 and Rab11 GTPase families direct lumen initiation. (a–e) Representative confocal microscopy images of an MDCK cyst at 48 h incubated with antibodies against the indicated proteins on stable expression of shRNA to knockdown Rab8a (b), Rab8b (c), Rab11a (d) and Rab25 (e). Control indicates stable expression of scrambled‑sequence shRNA (a). Bottom: higher‑magnification images of the indicated regions, showing podocalyxin localization (left), β‑catenin localization (middle), and a merge of these two images (right). Arrowheads indicate vesicular podocalyxin accumulation. (f) Proportion of single lumens in cysts with either stable knockdown or overexpression of dominant‑negative alleles (DN) of indicated Rab GTPases at 48 h, normalized with respect to control cells. Line represents 0.75‑fold single lumenogenesis. Values represent the mean ± s.d. of three or more experiments. Asterisk indicates P < 0.05 and triple asterisks indicate P < 0.0001. Control, n = 868; Rab8a, n = 302;

Rab8b, n = 312; Rab10, n = 336; Rab11a, n = 307; Rab11b, n = 1,528; Rab13, n = 315; Rab14, n = 331; Rab25, n = 310. (g, h) Representative images of a cyst expressing GFP–Rab11a (green) and incubated with Hoescht (blue) and with antibodies against Par3 (red) during lumen initiation. Bottom: higher‑magnification images of the indicated regions showing GFP–Rab11a (left), Par3 (middle) and a merge of these two images (right). Arrow (g) and arrowheads (h) indicate localization of Par3. (i–m) Experiments performed as in g and h except cells were incubated with antibodies against podocalyxin (red). In i; arrow indicates podocalyxin, and yellow and white arrowheads indicate localization of GFP–Rab11a to peripheral and juxtanuclear vesicles, respectively. In j, k; arrowhead indicates co‑localization of podocalyxin and GFP–Rab11a. In k–m; arrows indicate areas devoid of GFP–Rab11a, and arrowheads indicate clustering of GFP–Rab11a underneath the lumen. Scale bars, 20 μm.



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also mistargeted some GFP–CNT1 to cell–cell contacts (Supplementary Information, Fig. S3e). Moreover, on Par3 knockdown, Sec8 was not recruited to surface regions adjacent to vesicular podocalyxin (Fig. 4k), representing a failure to form the AMIS.

Inhibition of aPKC, using its pseudosubstrate inhibitor (aPKC-PS; Fig. 4m, n) similarly perturbed AMIS and single lumenogenesis,

causing accumulation of podocalyxin in Rab11a-positive vesicles, close to the surface marked by β-catenin (Fig. 4n; arrowheads). Additionally, aPKC inhibition caused lack of podocalyxin internalization from the periphery in some cells (Fig. 4n; arrows), probably representing an additional function of aPKC at this locale (see Fig. 1i). Together, these data demonstrate a crucial role for the exocyst–Par3–aPKC complex

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Figure 3 A Rab11–Rabin8–Rab8 complex governs apical transport and single lumenogenesis. (a) Rab11a recruits Rabin8 and Rab8a to subapical vesicles. Control MDCK cysts and cysts expressing GFP–Rab11a (wild‑type Rab11a; WT or an S25N substitution mutant; both green) were grown for 48 h and immunostained for either endogenous Rabin8 or Rab8a (both in red). There is diffuse vesicular labelling, with low‑level subapical accumulation of Rabin8 and Rab8a (arrows) in control cysts. Arrowheads indicate marked co‑recruitment and clustering of Rabin8 and Rab8a vesicles to the subapical region, which was absent in the GFP–Rab11aS25N mutant. (b) Knockdown of Rabin8 by two different shRNA were assessed by western blot analysis. Control cells stably expressed a scrambled‑sequence shRNA. GAPDH was used as a loading control. (c) Representative confocal microscopy image of an MDCK cyst at 48 h, stably expressing shRNA to knockdown Rabin8α, and incubated with anti‑podocalyxin antibodies, and Hoescht. Arrowheads indicate intracellular accumulation of podocalyxin. (d) Representative confocal images of MDCK cysts, stably

expressing shRNA to knockdown Rabin8α and expressing either RNAi‑resistant GFP–hRabin8α or a GFP– hRabin8αF201A mutant, were incubated with antibodies against podocalyxin and F‑actin. Arrows indicate luminal GFP–hRabin8αWT localization, and arrowheads indicate co‑localization of GFP– hRabin8αF201A and podocalyxin on vesicles. (e) Proportion of cysts with a single lumen in MDCK cysts stably expressing shRNA to knockdown Rabin8α, and expressing GFP–Rabin8αWT, GFP–Rabin8αL196A, or GFP–Rabin8αF201A, as indicated. Values represent the means ± s.d. of three or more experiments. Asterisk indicates P < 0.05, double asterisk indicates P < 0.001. Control, n = 312; Rabin8α KD, n = 340; Rabin8α KD + GFP–hRabin8αWT, n = 316; Rabin8α KD + GFP–hRabin8αL196A, n = 317; Rabin8α KD + GFP–hRabin8αF201A, n = 328. At the bottom of a and c and right of d are higher‑magnification images of the indicated regions showing localization of individual proteins (left, middle) and a merge of these two images (right). Scale bars, 20 μm. Uncropped images of blots are shown in Supplementary Information, Fig. S7a.



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Figure 4 The exocyst and Par3–aPKC regulate lumenogenesis. (a–c) Sec15A is required for AMIS formation. Representative images of a cyst at 48 h treated with Hoescht and with antibodies against podocalyxin, and β‑catenin (a, b) or expressing GFP–Rab11a (c). Cells were stably expressing either scrambled‑sequence shRNA (control) or shRNA specific to Sec15A. Bottom: higher‑magnification images of the indicated regions showing localization of individual proteins (left, middle) and a merge of these two images (right). Arrows (b) indicate localization of podocalyxin and arrowheads (c) indicate podocalyxin in vesicles labelled with GFP–Rab11a. (d) Proportion of cysts with a single lumen on stable expression of a control scrambled sequence shRNA or two different types of Sec15A shRNA, as indicated. Values represent the mean ± s.d. of three or more different experiments. Asterisk indicates P < 0.001. Control, n = 334; Sec15A_2, n = 355; Sec15A_5, n = 345. (e–h) Representative images of cysts at 48 h treated with Hoescht and antibodies against podocalyxin and Par3. Cells were stably expressing either scrambled sequence shRNA (control; e) or shRNA specific to Sec15A. In g, h the cysts are expressing an RNAi‑resistant GFP–Sec15A or a GFP–Sec15AN691A Rab‑uncoupled mutant. Par3 targeting to

the AMIS requires the exocyst. (e–h) Arrow indicates localization of Par3. (f, h) Arrowhead indicates podocalyxin vesicle coalescence. (i–k) The Par3–aPKC complex is required for lumen initiation. MDCK cells stably expressing shRNA specific to Par3 were imaged 48 h after plating. Arrows indicate podocalyxin localization. (l) Proportion of cysts with single lumens after knockdown of Par3 using two different shRNA, as indicated. Scrambled‑sequence shRNA was used as a control. Values represent the mean ± s.d. of three or more experiments. Asterisk indicates P

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in podocalyxin delivery from Rab11a-positive vesicles to form the lumen.

Annexin2–Cdc42 associates with Rab11a-positive vesicles during lumenogenesisLuminal targeting of aPKC in MDCK cysts requires interaction of GTP–Cdc42 with the phosphatidylinositol (4,5)-bisphosphate-binding protein, Annexin2 (Anx2)6. Anx2 both transits to the surface through, and regulates the function of, Rab11a recycling vesicles18,19. We thus examined the interaction between Anx2, Cdc42 and the Rab11a–Rab8a module.

In early cysts with peripheral podocalyxin (arrow), and subperiph-eral Apple–Rab11a, GFP–Anx2 localized to the surface (Supplementary Information, Fig. S6a). When podocalyxin was in condensed Rab11a-positive vesicles beneath the AMIS, some GFP–Anx2 now also localized to these vesicles (Supplementary Information, Fig. S6b; arrowheads). Once podocalyxin was at the open lumen (Supplementary Information, Fig. S6c; arrow), GFP–Anx2 localized to both apical and basolateral surfaces, but no longer to subapical Rab11a-positive vesicles. Thus, GFP–Anx2 transiently associates with Rab11a-positive vesicles during lumen initiation.

In contrast to controls expressing wild-type Anx2, which had luminal podocalyxin, subapical Apple–Rab11a, and apical and basolateral GFP–Anx2 (Supplementary Information, Fig. S6d, f), expression of dominant negative Anx2 (Anx2 XM) perturbed lumenogenesis and caused the accumulation of GFP–Anx2 and podocalyxin in Rab11a-positive vesi-cles (Supplementary Information, Fig. S6e; arrowheads). Conversely, knockdown of Rab8a or Rab11a caused intracellular accumulation of podocalyxin in structures co-labelled with GFP–Anx2 (Supplementary Information, Fig. S6g, h; arrowheads). Thus, Anx2 and Rab8a–Rab11a cooperate in the delivery of podocalyxin to the apical surface.

We next examined whether Cdc42 associated with Rab11a-positive vesicles. Unlike Anx2, GFP–Cdc42, although possessing a large cyto-plasmic pool, co-localized with subapical Apple–Rab11a in cysts with open lumens (Fig. 5a; arrowheads). Activated Cdc42 (GFP–Cdc42Q61L) localized to cell–cell contacts and the luminal region, marked by podo-calyxin (Fig. 5b; arrowheads). As GFP–Cdc42Q61L removed cytoplasmic background labelling, and expression did not perturb single lumenogen-esis (Fig. 6i), we used this allele to further examine Cdc42 localization. In early cysts with peripheral podocalyxin (arrows), GFP–Cdc42Q61L local-ized to the surface (Fig. 5c). When podocalyxin was internalized into Rab11a-positive vesicles and subsequently concentrated at the AMIS, GFP–Cdc42Q61L now extensively overlapped with these vesicles (Fig. 5d, e; arrowheads). As the PAP (Fig. 5f) and open lumen (Fig. 5g) formed, podocalyxin and Rab11a were no longer co-localized, whereas GFP–Cdc42Q61L maintained some overlap with both (arrows). Thus, active Cdc42 associates with Rab11a-positive vesicles during lumenogenesis.

Tuba–Cdc42 function in apical transport from Rab8a/Rab11a-positive vesiclesWe next determined whether Cdc42 is required for transport from Rab11a-positive vesicles. As shown previously6, Cdc42 knockdown perturbed lumenogenesis (Fig. 5n), and resulted in accumulation of podocalyxin in vesicular apical compartments (VACS; arrows) or vesicles (arrowheads) close to the surface marked by β-catenin (Fig. 5i). Notably, on Cdc42 knockdown, intracellular podocalyxin was localized to Rab8a/Rab11a-positive vesicles, suggesting Cdc42 regulates transport from these vesicles (Fig. 5l; arrowheads).

Intersectin 2 and Tuba have been identified as the only Cdc42-specific GEFs essential for MDCK lumenogenesis20,21. As intersectin-2 knock-down did not disrupt transport of podocalyxin in cysts20, we examined if Tuba regulates Cdc42-dependent podocalyxin transport. Tuba knock-down phenocopied Cdc42 knockdown, disrupted single lumenogenesis and accumulated podocalyxin in Rab8a/Rab11a-positive vesicles (Fig. 5j, m, n; arrowheads). Knockdown of Tuba or Cdc42 blocked the increase in single lumenogenesis induced by expression of GFP–Rab11a (Cdc42 had a larger effect; Fig. 5n), suggesting that Rab11a operates upstream of both Tuba and Cdc42. Thus, Tuba-dependent Cdc42 activation is required for podocalyxin apical transport.

Tuba is required for Cdc42 apical targeting21. We examined whether Rab8a or Rab11a also influenced Cdc42 activation. Knockdown of Rab8a, but not Rab11a, markedly decreased global GTP–Cdc42 levels (Fig. 6a). Similarly, overexpression of GFP–Rab8aQ67L, but not GFP–Rab11aQ70L, activated Cdc42 (Fig. 6b), suggesting that Rab8a influences global Cdc42 activation.

We examined whether Rab8a and Rab11a regulate apical Cdc42 tar-geting. A YFP-tagged p21-binding domain (PBD–YFP) probe of acti-vated Cdc42 (ref. 6) labelled the luminal surface, along with podocalyxin (Fig. 6c; arrowheads), and to a lesser extent cell–cell contacts, mirroring the localization of activated Cdc42 (Fig. 5b). Rab8a knockdown abro-gated PBD–YFP membrane association, despite retaining luminal podo-calyxin labelling (Fig. 6d; arrowhead). Strikingly, Rab11a knockdown resulted in a loss of apical (arrowhead), but not basolateral, PBD–YFP (arrows) (Fig. 6e). Thus, Rab8a is required for global activation and surface targeting of Cdc42, whereas Rab11a controls apical Rab8a, and consequently, active Cdc42 targeting.

We reasoned that as Rab8a and Rab11a influence apical targeting of active Cdc42, overexpression of active Cdc42 may rescue single lumeno-genesis on knockdown of Rab8a or Rab11a. Indeed, expression of active Cdc42 (GFP–Cdc42Q61L) rescued apical targeting of podocalyxin (Fig. 6g, h), and single lumenogenesis, in cysts with Rab8a or Rab11a knockdown (Fig. 6i). These data support the conclusion that Cdc42, regulated by Rab8a and Rab11a, is required for apical transport of podocalyxin. Therefore, Rab11a regulates a molecular network directing the apical polarity and trafficking machineries to initiate de novo lumen formation.

DISCUSSIONHow membrane trafficking and polarity-complex machineries work together to form the apical surface and lumen is a fundamental issue22. We describe a molecular chain linking membrane-trafficking machin-ery with delivery of the Par3–aPKC–Cdc42 complex to the nascent apical surface during lumen formation (Fig. 7a). This emphasizes the complex spatiotemporal orchestration needed to construct a new membrane (see Table 1).

Podocalyxin is initially localized to the extracellular matrix-contacting periphery (Fig. 7a), before internalization into Rab11a-positive vesicles, to which Rab8a is recruited through the GEF Rabin8 (which is inhibited by the GAP, TBC1D30; Fig. 7b). Apical vesicle delivery and lumenogen-esis is regulated by both Rab proteins. The exocyst, a Rab effector, docks vesicles with the apical surface to create the AMIS, in cooperation with Par3–aPKC (Fig. 7b). Anx2 and Cdc42 associate with Rab8a/Rab11a-positive vesicles, regulating apical transport and single lumenogenesis, dependent on the Cdc42 GEF, Tuba. Par6 probably bridges Cdc42 to the aPKC–Par3–exocyst complex at the AMIS. Thus, apical polarity and



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Figure 5 Tuba and Cdc42 regulate transport from Rab8a/Rab11a‑positive vesicles. (a–b) Representative images of 48 h cysts either co‑expressing Apple–Rab11a and GFP–Cdc42, and stained for nuclei (a), or expressing GFP–Cdc42Q61L and labelled for podocalyxin and nuclei (b). Arrowheads indicate co‑localization of Cdc42 and Rab11a for a, or localization of GFP–Cdc42Q61L and podocalyxin at the luminal surface for b. (c–g) Images of cysts expressing GFP–Cdc42Q61L at indicated stages of development. (c) Arrows indicate peripheral localization of podocalyxin. (d, e) Arrowheads indicate co‑localization of GFP–Cdc42Q61L and podocalyxin in Rab11a‑positive vesicles. (f, g) Arrows indicate localization of GFP–Cdc42Q61L and podocalyxin at the luminal surface. (h–j) Images of cysts at 48 h stably expressing shRNA specific to Cdc42 or Tuba. Cells were treated with Hoescht and with antibodies specific to podocalyxin and β‑catenin. (i, j) Arrow indicates accumulation of podocalyxin in VACS; arrowheads indicate podocalyxin localization in

vesicles. (k–m) Images of cysts at 48 h stably expressing GFP–Rab11a and shRNA specific to Cdc42 and Tuba, and treated with antibodies specific to podocalyxin and Rab8a. (k) Arrowheads indicate subapical GFP–Rab11a and Rab8a localization. (l, m) Arrowheads indicate co‑localization of podocalyxin, GFP–Rab11a and Rab8a in vesicles.Bottom (a–m): higher‑magnification images of the indicated regions showing localization of individual proteins and a merge of these images. Scale bars, 20 μm. (n) Proportion of MDCK cysts, and cysts expressing GFP–Rab11a, with a single lumen at 48 h after plating on stable expression of either shRNA specific to Cdc42 or Tuba, or a control scrambled‑sequence shRNA. Values represent the mean ± s.d. of three or more experiments. Double asterisks indicate P < 0.001, triple asterisks indicate P < 0.0001. Control, n = 311; Cdc42_2, n = 321; Cdc42_3, n = 307; Tuba_1, n = 319; Tuba_2, n = 319; GFP–Rab11a control, n = 600; GFP–Rab11a + Cdc42_2, n = 316; GFP–Rab11a + Tuba_1, n = 308.



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Figure 6 Rab8a/Rab11a regulate Cdc42 during apical transport. (a, b) Rab8a–Rab11a control Cdc42 activation and targeting. GTP–Cdc42 levels were assessed in MDCK cells stably expressing shRNA specific to Rab8a or Rab11a (a), or MDCK cells expressing activated Rab8a and Rab11a mutants (b), by pulldown and western blotting. Cell lysate was used to determine total Cdc42 levels. In a, graph represents GTP–Cdc42 levels normalized to total Cdc42 expression, and presented as a percentage of control cell levels of GTP–Cdc42. In b, these values are presented as a fold‑change, compared with control GTP–Cdc42 levels. Values represent the mean ± s.d. of three different experiments. Asterisk indicates P < 0.05. (c–e) Localization of a PBD–YFP probe to detect activated Cdc42 (and possibly also Rac) in MDCK cysts 48 h after plating. (c) Arrowheads indicate podocalyxin and PBD–YFP co‑localization. (d) Arrowheads indicate podocalyxin localization (which is not associated

with PBD–YFP when there is knockdown of Rab8a). (e) Arrowhead indicates loss of apical PBD–YFP and arrow indicates localization of PBD–YFP to the basolateral membrane. (f–h) Rescue of Rab8a/Rab11a knockdown by active Cdc42 overexpression in MDCK cysts at 48 h after plating. Arrowheads indicate apical targeting of GFP–Cdc42Q61L and podocalyxin. Bottom (c–h): higher‑magnification images of the indicated regions showing localization of individual proteins (left, middle) and a merge of these two images (right). Scale bar, 20 μm. (i) Effect of Rab8a and Rab11a knockdown on proportion of cysts with single lumens at 48 h after plating. Values represent means ± s.d. Asterisk indicates P < 0.05. Control, n = 868; Rab8a KD, n = 302; Rab11a KD, n = 307; GFP–Cdc42Q61L Control, n = 343; GFP–Cdc42Q61L + Rab8a KD, n = 319; GFP–Cdc42Q61L + Rab11a KD, n = 308. Uncropped images of blots are shown in Supplementary Information, Fig. S7a.

membrane domain identity is initiated de novo by membrane deliv-ery from Rab8a/Rab11a-positive vesicles. Finally, the luminal space is expanded by pumps and channels (Fig. 7b)7,23.

Podocalyxin is the earliest marker of apical polarization that we have studied, and its initial appearance at the nascent apical surface marks the AMIS. Notably, the Rab11a–Rabin8α–Rab8a cascade also regulates mam-malian ciliogenesis, and the homologous yeast pathway regulates bud-ding24, suggesting it is an ancient polarity-generating module.

The exocyst is a Rab effector required for transport of podocalyxin from Rab11a endosomes. This transport also requires the Par3–aPKC polar-ity complex, revealing a mutual interdependence of these complexes for localization to the AMIS. Similarly, in developing neurites, association of the exocyst and Par3–aPKC is required for Par3 localization to growing

neurite tips17. Interaction of the exocyst with Par3–aPKC may focus exocyst-dependent vesicle docking events transiently to the AMIS, allowing initia-tion of apical polarity, before both complexes relocalize to the tight junction for subsequent transport events. That the exocyst was also required for Par3 localization demonstrates that vesicle trafficking operates both upstream and downstream of cortical polarity proteins, suggesting a feedback loop.

Rab11a-positive vesicles also deliver the Crumbs3–Pals1–PatJ com-plex to early lumens25. In Drosophila, Crumbs functions to dissociate Par3 from Par6 and aPKC at the apical surface, restricting Par3 to api-colateral borders26,27. Similarly, Crumbs3 delivered to the AMIS may exclude a pool of Par3 from the nascent apical surface, allowing it to concentrate to the sides of the developing lumens, such as in the transi-tion from the AMIS to the PAP reported here.



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Par6 and aPKC form a complex with Cdc42 (ref. 28). Cdc42, in con-junction with Anx2 (ref. 6), regulates vesicular transport to the apical surface from Rab8a/Rab11a-positive vesicles. However, Cdc42 also regu-lates the orientation of cell division in conjunction with Par6–aPKC20,21,29. Indeed, the Cdc42 GEFs, intersectin 2 and Tuba, regulate Cdc42 acti-vation and orientation of division during MDCK lumenogenesis20,21. Intersectin-2 knockdown results in multiple lumens without vesicular podocalyxin accumulation20, suggesting it functions in cell division but not vesicle transport. In contrast, Tuba regulates apical targeting of Cdc42 (ref. 21) and, as demonstrated here, podocalyxin transport from Rab8a/Rab11a-positive vesicles.

Active Cdc42 localized to Rab11a-positive vesicles. In yeast, Cdc42 is also delivered to the bud site30, suggesting that vesicular transport of Cdc42 to membrane being generated de novo is a conserved polarity-generating event. Notably, Rab8a and Rab11a were required for Cdc42 activation at the lumen. Rab8a regulated global Cdc42 activation; Rab11a regu-lated apical Cdc42 targeting. That global Cdc42 activation was markedly decreased on Rab8a, but not Rab11a, knockdown suggests that, whereas Tuba functions downstream of Rab11a and regulates a pool of Cdc42 activation in Rab11a-positive vesicles, Rab8a may influence additional GEF proteins, such as intersectin 2. How Rab8a and Rab11a influence Cdc42 GEFs remains to be elucidated. Similarly, we demonstrate a role

for aPKC in apical transport from Rab11a-positive vesicles, suggesting that the Cdc42–Par6–aPKC complex may function in membrane trans-port, in addition to cell division31,32. These data reveal a novel role for Anx2–Cdc42–aPKC–Par3 in conjunction with the exocyst and Tuba in apical transport to the AMIS.

Our knockdown and overexpression experiments resulted in mul-tiple lumens and accumulation of apical proteins in subapical vesicles. Perturbation of apical traffic could cause multiple lumen formation through several overlapping mechanisms. Reduced apical delivery may prevent initially small lumens from enlarging and consolidating into one central lumen, which occurs in several mammalian organs33. Multiple small lumens also require ion pumping to hydrostatically enlarge and coalesce lumens. Apical trafficking defects could cause defective junctions and/or mislocalization of pumps and channels, preventing enlargement23.

Orientation of mitosis also regulates lumen formation. In Caco-2 cysts, Cdc42 knockdown correlates with disrupted spindle orientation, without notable apical transport defects29. Caco-2 lumen formation requires arti-ficially increasing cyclic AMP, which strongly promotes apical exocyto-sis34, potentially masking detection of apical transport defects. As Rab8a, Rab11a, exocyst and Par3–aPKC also have roles in spindle orientation and/or cytokinesis35–37, the extent to which their function in cell divi-sion is separate from apical trafficking is unknown. Apical membrane

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Figure 7 A molecular network for de novo lumen generation. (a) Schematic representation of the different stages of lumenogenesis and apical polarization in MDCK cysts. Initially, podocalyxin is localized to the periphery of cysts (early aggregate), before internalization into Rab8a and Rab11a‑positive vesicles, and delivery to the AMIS (apical membrane initiation). As podocalyxin at the apical domain and tight junctions become separately localized, the AMIS progresses to a PAP, representing the early stages of

apical–basal polarization. Expansion then allows opening of the luminal space. Note the co‑accumulation of polarization and trafficking machinery at the AMIS, despite varying localization during other stages of lumenogenesis. Red lines, podocalyxin; black lines, plasma membrane; grey ovals, nuclei; brown ovals, tight junctions; brown rectangle, AMIS; L, lumen. (b) A schematic representation of the molecular network involved in delivery of apical vesicles (podocalyxin) to the AMIS during apical membrane initiation.



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traffic might be needed to localize proteins that orient mitosis, such as Cdc42, aPKC and LGN (leucine-glycine-asparagine repeat-containing protein)20,21,38,39. Notably, multiple lumens can occur without disruption to apical transport20,39, suggesting that the two processes can be uncou-pled. For example, in MDCK and other systems, lumens can form in the absence of cell division or apoptosis7,40–43, instead requiring vesicular transport of podocalyxin to the cyst interior. Thus, coordination of divi-sion orientation and apical transport mechanisms are probably central in the generation of a single lumen.

Recently, the Par proteins, as well as aPKC and Cdc42, have been dem-onstrated as regulators of polarity through endocytosis32. We demon-strate that the membrane traffic, especially exocytosis, is both upstream and downstream of the Par complex. Our data support an emerging view of the Par complex as a multifunctional platform modulating membrane traffic31,32, and suggest Cdc42 –aPKC–Par3 as a convergence between the machineries of cortical polarization and vesicular transport.

METHODSMethods and any associated references are available in the online version of the paper at http://www.nature.com/naturecellbiology/

Note: Supplementary Information is available on the Nature Cell Biology website

ACKNOWLEDGEMENTSWe thank F. Barr, E. Brown, J. Stow, W. Guo, I. Macara, K. Simons, J. Wilson, T. Weimbs, and A. Zahraoui for gifts of reagents and unpublished data, and the Mostov lab for kind assistance. Supported by a Susan G Komen Foundation Fellowship (D.M.B.), a DOD Breast Cancer Concept Award (A.D.), NIH grants R01DK074398, R01AI25144 and P01AI53194 (K.E.M.), grants of the Human Frontiers Science Program (HFSP-CDA00011/2009), Marie Curie (IRG-209382), MICINN (BFU2008-01916) and (CONSOLIDER CSD2009-00016) to F.M.-B.; and a JAE fellowship (MICINN) to A.E.R.F.

AUTHOR CONTRIBUTIONSD.M.B., A.D., A.E.R.F, F.M.-B. and J.P. designed the experiments. D.M.B., A.D., A.E.R.F. and J.P. did the experimental work. D.M.B. and K.E.M. analysed the experiments. D.M.B. and K.E.M wrote the manuscript.

COMPETING FINANCIAL INTERESTSThe authors declare that they have no competing financial interests.

Published online at http://www.nature.com/naturecellbiologyReprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/

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M E T H O D S DOI: 10.1038/ncb2106

METHODSCyst culture. MDCK cysts were subcultured in 5% fetal bovine serum (FBS; Gibco), minimum essential medium (MEM) or grown in 3D Matrigel cultures (BD), as described6. Cells were trypsinized to a single cell suspension at 1.5 × 104 cells ml–1 in complete medium containing 2% Matrigel. Suspensions (250 μl) were plated into 8-well coverglass chambers (Nunc), pre-coated with 5 μl of 100% Matrigel. Cells were grown for 24–48 h before fixation in 4% paraformaldehyde (PFA). In some experiments, cells were treated with aPKC-PS (40 μM; Invitrogen) from the time of plating to inhibit aPKCs6. In most instances, exogenous proteins were from stably expressing cell lines. For Anx2 XM experiments, cells in 2D were transiently transfected with Anx2 XM (Lipofectamine 2000) 24 h before plating into 3D.

RNAi. Stable RNAi was achieved by viral shRNA. For Sec10 knockdown, cells were transfected with siRNA oligonucleotides, as previously described6. In all instances, knockdown was verified by western blot or quantitative real-time PCR (Q-PCR) procedures, normalized to GAPDH expression (Brilliant-II SYBR Green Kit, Agilent). Q-PCR primers are presented in Supplementary Information, Table S1. RNAi target sequences are presented in Supplementary Information, Table S2. Rab8a_1, Rab10, Rab11a_1 and Rab11b, in pRVH1-puro or –hygro retroviruses, were previously published44. All other shRNAs were generated in pLKO.1-puro45, or pLKO.1-blast, which was constructed by exchanging the puromycin resistance gene for blasticidin. Cdc42, Par3 and Tuba shRNAs were adapted for pLKO.1 from published sequences21,46. pLKO.1 lentiviruses were constructed according to the Addgene pLKO.1 protocol (www.addgene.org) using iRNAi (www.mekentosj.com), and target sequences were based on an (AA)N19 algorithm. RNAi sequences were submitted to BLAST (NCBI) to verify target specificity. For isoform-specific RNAi to Rabin8, shRNAs predicted to target the α isoform (Rabin8_4 or Rabin8_5) or to both α and β (Rabin8_2) isoforms of canine Rabin8 were extrapolated from sequence alignment with human Rabin8 splice forms (mined from NCBI). For knockdown and rescue experiments, GFP–tagged plasmids of transcripts from human or rat, which are not targeted by anti-canine shRNAs, were used.

Virus production and transduction. Retrovirus production was performed essentially as previously described44, except pRVH1 plasmids were transfected into 293-GPG cells (O. Weiner, UCSF)47. Post transfection (48 h), viral superna-tants were collected daily for 7 days. For lentivirus production, pLKO.1 plasmids were co-transfected with ViraPower packaging mix into 293-FT cells according to manufacturer’s instructions (Invitrogen). All supernatants were centrifuged to remove cell debris and frozen in liquid nitrogen for further use.

For retrovirus transduction (pRVH1), subconfluent cultures of MDCK cells, 16 h after plating, were incubated with virus-containing supernatants supple-mented with 10 μg ml–1 Polybrene (Millipore) for 24 h at 32 °C. On changing to fresh medium, cells were incubated for a further 24 h at 37 °C, before pas-sage into appropriate antibiotic-containing medium. For lentivirus transduction (pLKO.1), subconfluent MDCK cultures, 1–4 h after plating, were infected with virus-containing supernatants for 12–16 h at 37 °C. Viral supernatants were then diluted 1:1 with growth medium, cultured for a further 48 h, then passaged into appropriate antibiotic-containing medium. Hygromycin (0.5 mg ml–1), puromycin (5 μg ml–1), and blasticidin (12.5 μg ml–1) were used.

Plasmids and cell lines. Plasmids, the genes cloned into them and their source, and cell lines expressing the indicated genes and their source, were as follows: GFP–Cdc42 (used also for expression of the Q61L mutant; Addgene); GFP–Rab11a (used for expression of wild type and the S22N mutant; E. Brown, UCSF, USA); GFP–Rab8a (used for expression of wild type and the Q67L mutant) and GFP–Sec15A (J. Stow, University of Queensland, Australia); GFP–VSVG–podocalyxin and pRVH1-puro/hygro (used for expression of Rab8a, Rab10, Rab11a and Rab11b; K. Simons, EMBL, Germany); GFP–TBC1D30 (used for expression of wild type TBC1D30 and the R140A mutant; F. Barr, University of Liverpool, UK); GFP–Cdc42, YFP–PBD, GFP–Annexin2 (used for expression of wild type and XM)6 and GFP–Rab13 (used for expression of the T22N mutant; A. Zharaoui, CEA CNRS, France); GFP–Rab14 (used for the expression of the S25N mutant; J. Wilson, University of Arizona, USA); GFP–Rabin8α48 and Syntaxin3-2×myc (T. Weimbs, UCSD, USA)49 and GFP–CNT150. All additional plasmids were constructed through site-directed mutagenesis (Quikchange) or standard subcloning. For generation of stable lines, transfected cells underwent fluorescence activated cell sorting (FACS) after selec-tion to obtain appropriate expression levels.

Antibodies and immunolabelling. Primary mouse antibodies used were: anti-Cdc42 (1:1,000; BD Biosciences); anti-GAPDH (1:10,000; Millipore, Billerica, MA); anti-gp135/podocalyxin (1:1,000; G. Ojakian, SUNY, USA); anti-myc (1:200; 9E10, Santa Cruz Biotechnology); anti-p58 and anti-Na+/K+-ATPase (1:200; K. Matlin); anti-Tuba (1:500; Abnova) and anti-VSVG (1:1,000; P5D4)51. Primary rabbit antibodies used were: anti-aPKCζ (1:200; C-20) and anti-β-catenin (1:200; Santa Cruz Biotechnology); anti-GFP (1:1,000; Invitrogen); anti-occludin (1:200; Invitrogen); anti-Par3 (Immunofluorescence, 1:100, west-ern blot, 1:1,000; Millipore); anti-Rabin8 (immunofluorescence, 1:50; western blot, 1:500; ProteinTech); anti-Rab8a (immunofluorescence, 1:50, western blot, 1:500)48; anti-Rab8b (1:500; ProteinTech); anti-Rab10 (1:500; Sigma); anti-Rab11a (immunofluorescence, 1:100, western blot, 1:1,000; Millipore); anti-Rab25 (1:500; Cell Signaling Technology); anti-Sec8 (1:100; Enzo Life Sciences); anti-Sec10 (immunofluorescence, 1:250 in methanol:acetic acid, western blot, 1:250; W. Guo, University of Pennsylvania, USA). Rat anti-ZO-1 (1:200; R40.76, B. Stevenson) was also used. Alexa fluorophore-conjugated secondary antibodies (1:250 for all secondary antibodies) or Phalloidin (1:400; Invitrogen) and Hoescht to label nuclei (10 μg ml–1), were utilized. Cysts were stained as previously described6 .

Statistics. Single lumen formation was quantified as previously described6. The percentage of cysts with a single lumen was determined, and normalized to control cysts. Values are mean ± s.d. from three replicate experiments, with n ≥100 cysts per replicate. Significance was calculated using a paired, two-tailed Student’s t-test.

Transcytosis and IgA uptake. For podocalyxin transcytosis, cells stably express-ing GFP–VSVG–podocalyxin were plated into 8-well chamber slides and grown for 12 h at 37 °C. Slides were placed on ice and washed twice with serum-free medium at 4 °C before incubation at 4 °C for 30 min with serum-free medium containing the indicated antibodies. Cysts were then washed twice with cold serum-free medium, before being grown for 24 h at 37 °C in fresh serum-con-taining medium supplemented with 2% Matrigel.

Polymeric immunoglobulin A (pIgA), provided by J. P. Vaerman (Catholic University of Louvain, Belgium), was biotinylated using sulfo-NHS-LC-biotin (Pierce). For IgA uptake, MDCK PTR-9 cells stably expressing human TfR and rabbit polymeric immunoglobulin receptor (pIgR)52 were grown for 36 h in 3D to induce cyst formation, then incubated with 100 μg ml–1 biotinylated IgA in complete medium for 40 min. Biotinylated IgA was detected using fluorescently labelled streptavidin (Invitrogen).

GTPase activation. GTP loading of Cdc42 was by GST-PAK pull down (Cytoskeleton, Denver), according to manufacturer’s instructions, with modifi-cations. Cells were lysed in Mg Lysis Buffer (Millipore) containing 1 mM PMSF (phenylmethanesulfonylfluoride) and protease inhibitor cocktail, and lysates were passed through a 27.5 gauge needle (BD), and cleared of debris by centrifugation. A sample of lysate was taken for protein concentration determination (BCA), and lysates were snap-frozen. Protein (3 μg) from the appropriate condition was incubated with 20 μg of GST-PAK bead for 30 min at 4 °C. Beads were collected by centrifugation and washed three times in buffer before SDS–polyacrylamide gel electrophoresis (SDS–PAGE). Total Cdc42 and GTP–Cdc42 levels were detected by western blotting using an anti-Cdc42 antibody (BD Biosciences).

Rabin8 mutagenesis. GEF mutants of Rabin8 were modelled on critical, con-served residues in the Rab GEF domain governing Sec2p–Sec4p interactions53. L196A and F201A mutants were generated from pEGFP-C1-Rabin8α and pGEX-2T-Rabin8α48 (Quikchange). Decreased, direct association of Rab8a with Rabin8 mutants was verified by GST pulldown. Briefly, GST–Rabin8, GST–Rabin8L196A, GST–Rabin8F201A (15 °C overnight; 200 μM IPTG) or GST protein expression (37 °C for 3 h) was induced in bacteria. Lysates containing GST fusion proteins were incu-bated with glutathione–agarose beads (Sigma) at 4 °C for 2 h, then washed three times during 30 min with binding-buffer (50 mM Tris at pH 7.5, 150 mM NaCl, 2 mM MgCl2 and 1% Triton X-100). Rab8A

T22N and Rab8AQ67L were translated in vitro using a TNT Quick kit (Promega) according to manufacturer’s instruc-tions. The in vitro translation products were incubated with GST proteins coupled to glutathione–agarose beads in binding buffer (50 mM Tris at pH 7.5, 150 mM NaCl, 2 mM MgCl2 and 1% Triton X-100) on a rotating wheel at 4 °C for 1 h. The beads were washed four times with binding buffer over 30 min. Bound material was eluted from the beads with Laemmli sample buffer and loaded onto a 12%

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DOI: 10.1038/ncb2106 M E T H O D S

SDS–PAGE gel. As a control, 1/10 of the in vitro translation reactions were used. Bands were visualized by autoradiography of the dried gels. Densitometry revealed that the L196A and F201A mutants decreased association with Rab8a by 39% and 52%, respectively, confirming their reduction-of-function characteristics.

44. Schuck, S., Manninen, A., Honsho, M., Fullekrug, J. & Simons, K. Generation of single and double knockdowns in polarized epithelial cells by retrovirus‑mediated RNA interference. Proc. Natl Acad. Sci. USA 101, 4912–4917 (2004).

45. Moffat, J. et al. A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high‑content screen. Cell 124, 1283–1298 (2006).

46. Sfakianos, J. et al. Par3 functions in the biogenesis of the primary cilium in polarized epithelial cells. J. Cell Biol. 179, 1133–1140 (2007).

47. Ory, D. S., Neugeboren, B. A. & Mulligan, R. C. A stable human‑derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes. Proc. Natl Acad. Sci. USA 93, 11400–11406 (1996).

48. Hattula, K., Furuhjelm, J., Arffman, A. & Peranen, J. A Rab8‑specific GDP/GTP exchange factor is involved in actin remodeling and polarized membrane transport. Mol. Biol. Cell 13, 3268–3280 (2002).

49. Kreitzer, G. et al. Three‑dimensional analysis of post‑Golgi carrier exocytosis in epithelial cells. Nat. Cell Biol. 5, 126–136 (2003).

50. Mangravite, L. M., Lipschutz, J. H., Mostov, K. E. & Giacomini, K. M. Localization of GFP‑tagged concentrative nucleoside transporters in a renal polarized epithelial cell line. Am. J. Physiol. Renal. Physiol. 280, F879–F885 (2001).

51. Kreis, T. E. Microinjected antibodies against the cytoplasmic domain of vesicular sto‑matitis virus glycoprotein block its transport to the cell surface. Embo J. 5, 931–941 (1986).

52. Brown, P. S. et al. Definition of distinct compartments in polarized Madin‑Darby canine kidney (MDCK) cells for membrane‑volume sorting, polarized sorting and apical recycling. Traffic 1, 124–140 (2000).

53. Sato, Y. et al. Asymmetric coiled‑coil structure with guanine nucleotide exchange activity. Structure 15, 245–252 (2007).

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s u p p l e m e n ta ry i n f o r m at i o n

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DOI: 10.1038/ncb2106

Figure S1 Localization of apical and basolateral proteins during cystogenesis. (a-f) Early apical polarization is a feature of podocalyxin in cysts. MDCK cysts stably expressing GFP-podocalyxin (a,d), Syntaxin-3-2xmyc (b,e), or GFP-CNT1 (c,f) (all green) were co-stained for nuclei (nuc, blue), and either Na/K-ATPase (red, a,d), or podocalyxin (red, c,f) 12 h after plating (a-c) or once lumens had formed (48 h, d-f). Note that GFP-podocalyxin, but not Syntaxin-3 or GFP-CNT1 are excluded from cell-cell contacts in early aggregates. Smaller panels (c,f, right) depict higher magnification images of regions indicated. Arrowheads, GFP-CNT1 at cell-cell contacts. (g) Podocalyxin transcytoses to the lumen. Binding of antibodies (α-myc,

α-VSVG, α-GFP; red) to cysts expressing GFP-VSVG-podocalyxin (green) at 4°C, followed by allowing lumen development to occur revealed transcytosis of α-VSVG and α-GFP, but not non-specific α-myc antibodies, with podocalyxin from the periphery to the PAP (arrowheads), and to intracellular vesicles (arrows). Yellow, colocalization of podocalyxin and bound antibodies. (h) Lumens are accessible to transcytosed IgA. MDCK cysts stably expressing pIgR were grown for 36 h, and incubated with 100 µg/ml biotinylated IgA (IgA) for 40 min to allow transcytosis. Cells were stained for Rab11a (green), nuclei (blue), and IgA using fluorescent streptavidin (red). Note IgA transcytosed to the PAP (arrow). Bar, 20 µm.

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Figure S2 Sec10 analysis during lumen formation. (a-d) Sec10 labeling condenses towards the AMIS and tight junctions. During lumenogenesis, Sec10 (red) initially localizes along the cell-cell contact (a) when podocalyxin (green) is peripheral, then condenses towards the AMIS, overlapping with podocalyxin (b), before redistributing to sub-luminal puncta (c,d). (e-f) Sec10 localizes to the tight junctions once lumens form. 72h MDCK cysts were labeled for Sec10 (red), ZO-1 (blue), and either podocalyxin (e) or β-catenin (f) (both in green). Arrowheads, Sec10 and ZO-1 at tight junction regions. (g-i) Knockdown of Sec10 disrupts single lumenogenesis. Lysates of MDCK transfected with siRNAs against Sec10

alone (1, 2, 3), in combination (pool), or with control siRNAs were blotted for Sec10 and actin as a loading control (g). In contrast to control cysts (h) with luminal podocalyxin (red, arrowhead) and subapical Sec10 puncta (green), Sec10 knockdown cysts lost Sec10 labeling, did not form a single lumen, and showed vesicular accumulation of a pool of podocalyxin (arrows). Sec10 knockdown significantly reduced single lumenogenesis (i). Lumenogenesis quantitation values represent the average of ≥ three different experiments ± S.D., where *p



Figure S3 Characterization of MDCK RNAi. (a-e) Apical exocytosis machinery kncockdown disrupts lumen formation in cysts. MDCK GFP-CNT1 (green) cysts stably expressing control (a), Rab11a (b; Rab11a_2), Rab8a (g; Rab8a_2), Sec15A (d; Sec15A_2), or Par3 (e; Par3_4) shRNAs were grown for 48 h and labeled for Na/K-ATPase (red) and nuclei (blue). Note apical GFP-CNT1, and basolateral Na/K-ATPase, localization in control cysts, and disruption to GFP-CNT1 localization, but not Na/K-ATPase, localization in apical exocytosis/polarity machinery. Arrowheads, GFP-CNT1 at cell-cell contacts. (f-k, m-p) Validation of shRNAs. Knockdown with (f) primary Rab8a and Rab11a shRNAs alone or in combination, (g) secondary Rab8a and Rab11a shRNAs, (h) both Rab8b shRNAs, (i) Rab10 shRNA, (j) primary Rab11a and Rab11b shRNA, (k) both Rab25 shRNA, (m) both Sec15A shRNA, (n) both Par3 shRNA, (o) both Cdc42 shRNA, and (p) both Tuba

shRNA was verified by blotting with appropriate antibodies (f-i, k, n-p), or by qRT-PCR (j, m), with GAPDH used as a control in all instances. For qRT-PCR, values are mean ± SD from three replicates. *p < 0.05. (l) Validation of multiple Rab8/11 family member shRNAs on single lumen formation. Single lumen formation in 48 h cysts expressing indicated shRNA to Rab8 and Rab11 family members was quantified, confirming that multiple shRNAs targeting Rab8a, Rab8b, Rab11a, and Rab25 all disrupt single lumenogenesis. Line represents 0.75-fold single lumenogenesis, normalized to control levels. Values represent the average of ≥ three different experiments ± S.D., where **p



Figure S4 Additional Rab GTPase analysis during lumen formation. (a) Rab11a promotes lumen formation. Quantitation of single lumenogenesis at 48 h in control cysts or cysts stably expressing GFP-Rab11a (WT, S25N, or Q70L) revealed that GFP-Rab11a WT or Q70L expression significantly increased single lumenogenesis compared to control (MDCK alone), while the S25N mutant strongly suppressed single lumenogenesis. Values represent the average of ≥ three different experiments ± S.D., where **p



Figure S5 Characterization of Rab8 regulatory proteins. (a) Cartoon diagram of Rabin8 isoforms, and conserved, critical guanine nucleotide exchange activity residues. Amino acid positions refer to human Rabin8α. RBR, Rab11-binding region; Cf, canis familiaris; hs, homo sapiens; rn, rattus norvegicus; sc, saccharomyces cerevisiae. Boxed residues indicate crucial residues for GEF activity. (b-c) Characterization of Rab8a binding to Rabin8α. In vitro binding of Rab8a-T22N to recombinant GST-Rabin8α WT, GST-Rabin8α-L196A, GST-Rabin8α-F201A and GST, and Rab8a-Q67L to GST-Rabin8α WT and GST-Rabin8α-L196A (b), as described in Methods. Lanes at right indicate 1/10 of input of in vitro translated Rab8a-T22N and Rab8a-Q67L. Corresponding GST-proteins from beads used for the binding assays were run on SDS-PAGE and stained with Coomassie blue. Densitometry revealed that the L196A

and F201A mutants decreased association with Rab8a-T22N by 39% and 52%, respectively, confirming their reduction-of-function characteristics (c). Note negligible association of any Rabin8α allele with Rab8a-Q67L. (d-e) TBC1D30 GAP activity inhibits single lumen formation. MDCK cysts stably expressing WT or GAP-deficient (R140A) GFP-TBC1D30 (green) grown for 48 h were costained for podocalyxin (red) and nuclei (blue). Quantitation (e) revealed that GFP-TBC1D30 strongly suppressed single lumenogenesis, and that this required its GAP activity (i.e. R140A mutant did not perturb single lumenogenesis). Lumenogenesis quantitation values represent the average of ≥ three different experiments ± S.D., where *p



Figure S6 Annexin2 associates with Rab11a vesicles. (a-c) Anx2 transiently associates with Rab11a vesicles. Examination of cysts expressing GFP-Anx2 (green), Apple-Rab11a (red), and labelled for podocalyxin (blue), revealed that Anx2 localized to Rab11a vesicles (arrowheads) during lumen initiation (b, AMIS), but not when podocalyxin was peripheral (a), or once lumens formed (c, luminal podocalyxin, arrows). (d-e) Dominant negative Anx2 (Anx2 XM) disrupts single lumenogenesis. In control cysts (d) GFP-Anx2 (green) was at the apical and basolateral surface, Apple-Rab11a (pseudo-coloured blue) was in subapical vesicles,

and podocalyxin (red) was at the lumen. In contrast, expression of Anx2 XM disrupted single lumenogenesis and caused the accumulation of a pool of podocalyxin and Anx2 in Apple-Rab11a vesicles (e, arrowheads). (f-h) Rab8a/11a knockdown accumulates a pool of intracellular Anx2. In contrast to control cysts (f) with luminal podocalyxin (red) and apical and basolateral GFP-Anx2 (green), Rab8a (g) or Rab11a (h) knockdown perturbed lumenogenesis and co-accumulated Anx2 and podocalyxin in intracellular vesicles (arrowheads). Blue, nuclei. Smaller panels indicate higher magnification of regions indicated. Bar, 20 µm.

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Figure S7 Full scans of Western blots.

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Supplementary Table 1: Q-RT PCR Primer sequences. Target Primer Sequence (5’ – 3’) GAPDH Fwd AGTCAAGGCTGAGAACGGGAAACT Rev CATGGTTCACGCCCATCACAAACA Rab11a Fwd GCATCCAGGTTGATGGGAAA Rev AGGCACCTACAGCTCCACGA Rab11b Fwd GCTGGTGGGCAACAAGAGTG Rev GGTGGAATCCAAGGCTGAGG Sec15A Fwd GTCAGCCTGCCAGCATCTGT Rev CTGCTGAACAGCTCCCATGC


Supplementary Table 2: RNAi target sequences Target Vector Sequence (5’ – 3’) Control pLKO.1-puro CCGCAGGTATGCACGCGT Cdc42_2 pLKO.1-blast GATTACGACCGCTGAGTTA Cdc42_3 pLKO.1-blast GCGATGGTGCCGTTGGTAA Par3_3 pLKO.1-puro GACATCATGAAAGCTAGAA Par3_4 pLKO.1-puro GAGTATGGAGAGGCACATC Rab8a_1 pRVH1-puro/hygro (AA)GACAAGTTTCCAAGGAACG Rab8a_2 pLKO.1-puro GGGCCCTCCCCCTCCAATACT Rab8b_1 pLKO.1-blast (AA)GCGGGCAGAGCCAGGAATT Rab8b_3 pLKO.1-blast (AA)GAGGAGAGAAGTTAGCAAT Rab10 pRVH1-puro (AA)GCTGAAGATATCCTTCGAAAG Rab11a_1 pRVH1-puro/hygro (AA)GGCACAGATATGGGACACA Rab11a_2 pLKO.1-puro (AA)GGTTTGTCATTCATTGAG Rab11b pRVH1-puro (AA)GAACATCCTCACAGAGATC Rab25_2 pLKO.1-blast (AA)AGAGATCTTCACCAAAGTG Rab25_4 pLKO.1-blast (AA)CCAGGCGCTGGTCAGAAGA Rabin8_4 pLKO.1-puro (AA)GCCCTGTAAACACAGAATT Rabin8_5 pLKO.1-puro (AA)GCTGGGATATTTCAAAGAG Sec10_1 siRNA GCACATCAGCTATGTAGCAACCAA Sec10_2 siRNA CAGATCCTCCTGATTTACCAAGGAT Sec10_3 siRNA CCAAAGACTTCAAGATTCCACTGGTA Sec15A_2 pLKO.1-puro (AA)GAGGATGAGAATGAAGAGG Sec15A_5 pLKO.1-puro (AA)GCACGGGTCATGATAGTTT Tuba_1 pLKO.1-blast GGAAATATGCAGATGGTGA Tuba_2 pLKO.1-blast GCAGAGAAGTTAAAGGACA


A molecular network for de novo generation of the apical surface and lumenDISCUSSIONACKNOWLEDGEMENTSAUTHOR CONTRIBUTIONSReferencesMETHODSFigure 1 Characterization of lumen initiation in MDCK cysts. (a) Representative immunofluorescence confocal microscopy images showing development of polarity in MDCK cysts incubated with antibodies against the indicated proteins, and Hoescht (to visualize nuclei). Arrow at 24 h after plating indicates polarity inversion, with podocalyxin now at the cyst interior and formation of a PAP. Arrow at 48 h after plating indicates the opening of the luminal space. (b) Schematic representation of cyst development from a. Black lines, plasma membrane; red lines, apical surface; blue, nuclei. (c–k) Transient localization of polarity and trafficking machinery to an apical membrane initiation site (AMIS) in representative MDCK cysts at different stages during lumen initiation. Bottom: higher-magnification images of the indicated regions showing localization of individual proteins (c–e), or in f–k individual proteins (left, middle) and a merge of these two images (right). Arrowheads in c–e indicate Sec8 and Par3 localization. Arrow in d indicates co-localization of Sec8, Par3 and GFP–podocalyxin (green) at the AMIS. Arrows in f indicate co-localization of podocalyxin and occludin at the AMIS; arrowheads in g and h indicate localization of occludin at the edges of the lumen (g) and at the tight junctions (h). In i arrowheads indicate peripheral podocalyxin and aPKC. Arrows in i indicate localization of aPKC at the AMIS. In j–k arrowheads indicate aPKC at tight junction regions. Scale bars, 20 μm. Figure 2 The Rab8 and Rab11 GTPase families direct lumen initiation. (a–e) Representative confocal microscopy images of an MDCK cyst at 48 h incubated with antibodies against the indicated proteins on stable expression of shRNA to knockdown Rab8a (b), Rab8b (c), Rab11a (d) and Rab25 (e). Control indicates stable expression of scrambled-sequence shRNA (a). Bottom: higher-magnification images of the indicated regions, showing podocalyxin localization (left), β-catenin localization (middle), and a merge of these two images (right). Arrowheads indicate vesicular podocalyxin accumulation. (f) Proportion of single lumens in cysts with either stable knockdown or overexpression of dominant-negative alleles (DN) of indicated Rab GTPases at 48 h, normalized with respect to control cells. Line represents 0.75-fold single lumenogenesis. Values represent the mean ± s.d. of three or more experiments. Asterisk indicates P

a molecular network for de novo generation of the apical ......to form epithelial organs cells must...

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